The step-by-step evolution of novel phenotypes is central to several fundamental questions in biology. In studies of novel protein functions, the problem becomes experimentally tractable if it is possible to identify and functionally characterize the complete set of causative mutations. With such a system, it is possible to address key questions: Do novel functions evolve via the successive fixation of beneficial mutations that each produce an adaptive change in phenotype when they first arise? Alternatively, are evolutionary transitions in protein function facilitated by neutral mutations that produce no adaptive benefit when they first arise, but which potentiate the function-altering effects of subsequent mutations? By reconstructing all possible mutational pathways that connect ancestral and descendant proteins it is also possible to address fundamental questions about the roles of contingency and determinism in protein evolution. For example: Can novel functions evolve from any possible ancestral starting point, or are specific evolutionary outcomes contingent on prior history? We will address these questions by experimentally dissecting the molecular basis of a key physiological innovation during vertebrate evolution. Specifically, we will examine the evolution of a unique allosteric mechanism for regulating hemoglobin (Hb) function in the red blood cells of crocodilians. This unique mode of allosteric regulatory control contributes to crocodilians? extraordinary capacities for breath-hold diving. Using ancestral protein resurrection in conjunction with a combinatorial protein engineering approach based on site- directed mutagenesis, we will examine the effects of sequential mutational steps in the evolution of the novel allosteric mechanism of crocodilian Hb. We will also obtain insights into the structural basis of the change in Hb function, as X-ray crystallography experiments will reveal biophysical mechanisms at atomic resolution. The specific aims of the project are as follows: (1) Identify the specific mutations that are responsible for the evolution of the novel protein function, and quantify their additive and nonadditive effects; and (2) Identify and characterize the biophysical mechanisms responsible for the functional transition (gain of novel function, loss of ancestral function). In combination, accomplishing Specific Aims 1 and 2 will reveal the molecular basis of a key physiological innovation and will provide general insights into the pathways by which such innovations evolve.